John M. Bekkers

Detection of spontaneous synaptic events with an optimally scaled template

Spontaneous synaptic events can be difficult to detect when their amplitudes are close to the background noise level. Here we report a sensitive new technique for automatic detection of small asynchronous events. A waveform with the time course of a typical synaptic event (a template) is slid along the current or voltage trace and optimally scaled to fit the data at each position. A detection criterion is calculated based on the optimum scaling factor and the quality of the fit. An event is detected when this criterion crosses a threshold level. The algorithm automatically compensates for changes in recording noise. The sensitivity and selectivity of the method were tested using real and simulated data, and the influence of the template parameter settings was investigated. Its performance was comparable to that obtained by visual event detection, and it was more sensitive than previously described threshold detection techniques. Under typical recording conditions, all fast synaptic events with amplitudes of at least three times the noise standard deviation (3 sigma) could be detected, as could 75% of events with amplitudes of 2 sigma. The scaled template technique is implemented within a commercial data analysis application and can be applied to many standard electrophysiological data file formats.

Apical Dendritic Location of Slow Afterhyperpolarization Current in Hippocampal Pyramidal Neurons: Implications for the Integration of Long-Term Potentiation

Trains of action potentials in hippocampal pyramidal neurons are followed by a prolonged afterhyperpolarization (AHP) lasting several seconds, which is attributable to the activation of a slow calcium-activated potassium current (sIAHP). Here we examine the location of sIAHP on CA1 pyramidal neurons by comparing it with two GABAergic inhibitory postsynaptic currents (IPSCs) with known somatic and dendritic locations. Whole-cell patch-clamp recordings were made from CA1 pyramidal neurons in acute hippocampal slices. Stepping the membrane potential at the peak of sIAHP produced a relaxation (“switchoff”) of the AHP current with a time constant of 7.4 ± 0.4 msec (mean ± SEM). The switchoff time constants for somatic and dendritic GABAA IPSCs were 3.5 ± 0.5 msec and 8.8 ± 0.3 msec, respectively. This data, together with cable modeling, indicates that active sIAHP channels are distributed over the proximal dendrites within ∼200 μm of the soma. Excitatory postsynaptic potentials (EPSPs) evoked in stratum (s.) radiatum had their amplitudes shunted more by the AHP than did EPSPs evoked in s. oriens, suggesting that active AHP channels are restricted to the apical dendritic tree. Blockade of the AHP during a tetanus, which in control conditions elicited a decremental short-term potentiation (STP), converted STP to long-term potentiation (LTP). Thus, activation of the AHP increases the threshold for induction of LTP. These results suggest that in addition to its established role in spike frequency adaptation, the AHP works as an adjustable gain control, variably hyperpolarizing and shunting synaptic potentials arising in the apical dendrites.

Distribution and activation of voltage‐gated potassium channels in cell‐attached and outside‐out patches from large layer 5 cortical pyramidal neurons of the rat

1 Voltage‐gated potassium channels were studied in cell‐attached and outside‐out patches from the soma and primary apical dendrite of large layer 5 pyramidal neurons in acute slices of rat sensorimotor cortex (22–25 °C). 2 Ensemble averages revealed that some patches contained only fast, IA‐like channels, others contained only IK‐like channels that did not inactivate or inactivated slowly, and the remainder contained mixtures of both channel types. IA and IK channels had mean unitary conductances of 8.5 and 20.3 pS, respectively, and had distinctive patterns of gating. 3 Peak activation curves for ensemble‐averaged currents were described by the Boltzmann equation with half‐maximal voltage (V½) and slope factor (k) values of −24.5 mV and 16.9 mV for IA and −7.6 mV and 10.1 mV for IK (patches < 250 μm from the soma) or −22.9 mV and 16.2 mV for IA (patches > 250 μm from the soma). The steady‐state inactivation curve for IA gave V½ and k values of −72.3 mV and −5.9 mV (< 250 μm from the soma) or −83.1 mV and −6.5 mV (> 250 μm from the soma). These values were similar to the corresponding data for IA and IK in nucleated patches from the same cell type. 4 The amount of IA and IK present in patches depended weakly on distance along the primary apical dendrite from the soma. The amplitude of IA increased, on average, by 2.3 pA per 100 μm, while the amplitude of IK decreased by 0.4 pA per 100 μm. 5 I A and IK channels in dendritic cell‐attached patches were activated by the passage of a back‐propagating action potential past the tip of the patch electrode. These results show directly that these potassium channels participate in action potential repolarisation, and thus contribute to the process of synaptic integration in these neurons.

Properties of voltage-gated potassium currents in nucleated patches from large layer 5 cortical pyramidal neurons of the rat

1 Voltage‐gated potassium currents were studied in nucleated outside‐out patches obtained from large layer 5 pyramidal neurons in acute slices of sensorimotor cortex from 13‐ to 15‐day‐old Wistar rats (22–25 °C). 2 Two main types of current were found, an A‐current (IA) and a delayed rectifier current (IK), which were blocked by 4‐aminopyridine (5 mm) and tetraethylammonium (30 mm), respectively. 3 Recovery from inactivation was mono‐exponential (for IA) or bi‐exponential (for IK) and strongly voltage dependent. Both IA and IK could be almost fully inactivated by depolarising prepulses of sufficient duration. Steady‐state inactivation curves were well fitted by the Boltzmann equation with half‐maximal voltage (V½) and slope factor (k) values of −81.6 mV and −6.7 mV for IA, and −66.6 mV and −9.2 mV for IK. Peak activation curves were described by the Boltzmann equation with V½ and k values of −18.8 mV and 16.6 mV for IA, and −9.6 mV and 13.2 mV for IK. 4 I A inactivated mono‐exponentially during a depolarising test pulse, with a time constant (∼7 ms) that was weakly dependent on membrane potential. IK inactivated bi‐exponentially with time constants (∼460 ms, ∼4.2 s) that were also weakly voltage dependent. The time to peak of both IA and IK depended strongly on membrane potential. The kinetics of IA and IK were described by a Hodgkin‐Huxley‐style equation of the form mNh, where N was 3 for IA and 1 for IK. 5 These results provide a basis for understanding the role of voltage‐gated potassium currents in the firing properties of large layer 5 pyramidal neurons of the rat neocortex.

Quantal amplitude and quantal variance of strontium-induced asynchronous EPSCs in rat dentate granule neurons

1 Excitatory postsynaptic currents (EPSCs) were recorded from granule cells of the dentate gyrus in acute slices of 17‐ to 21‐day‐old rats (22‐25 °C) using tissue cuts and minimal extracellular stimulation to selectively activate a small number of synaptic contacts. 2 Adding millimolar Sr2+ to the external solution produced asynchronous EPSCs (aEPSCs) lasting for several hundred milliseconds after the stimulus. Minimally stimulated aEPSCs resembled miniature EPSCs (mEPSCs) recorded in the same cell but differed from them in ways expected from the greater range of dendritic filtering experienced by mEPSCs. aEPSCs had the same stimulus threshold as the synchronous EPSCs (sEPSCs) that followed the stimulus with a brief latency. aEPSCs following stimulation of distal inputs had a slower mean rise time than those following stimulation of proximal inputs. These results suggest that aEPSCs arose from the same synapses that generated sEPSCs. 3 Proximally elicited aEPSCs had a mean amplitude of 6.7 ± 2.2 pA (± s.d., n= 23 cells) at ‐70 mV and an amplitude coefficient of variation of 0.46 ± 0.08. 4 The amplitude distributions of sEPSCs never exhibited distinct peaks. 4 Monte Carlo modelling of the shapes of aEPSC amplitude distributions indicated that our data were best explained by an intrasite model of quantal variance. 5 It is concluded that Sr2+‐evoked aEPSCs are uniquantal events arising at synaptic terminals that were recently invaded by an action potential, and so provide direct information about the quantal amplitude and quantal variance at those terminals. The large quantal variance obscures quantization of the amplitudes of evoked sEPSCs at this class of excitatory synapse.

Targeted dendrotomy reveals active and passive contributions of the dendritic tree to synaptic integration and neuronal output

Neurons typically function as transduction devices, converting patterns of synaptic inputs, received on the dendrites, into trains of output action potentials in the axon. This transduction process is surprisingly complex and has been proposed to involve a two-way dialogue between axosomatic and dendritic compartments that can generate mutually interacting regenerative responses. To manipulate this process, we have developed a new approach for rapid and reversible occlusion or amputation of the primary dendrites of individual neurons in brain slices. By applying these techniques to cerebellar Purkinje and layer 5 cortical pyramidal neurons, we show directly that both the active and passive properties of dendrites differentially affect firing in the axon depending on the strength of stimulation. For weak excitation, dendrites act as a passive electrical load, raising spike threshold and dampening axonal excitability. For strong excitation, dendrites contribute regenerative inward currents, which trigger burst firing and enhance neuronal excitability. These findings provide direct support for the idea that dendritic morphology and conductances act in concert to regulate the excitability of the neuron.

Neural Coding by Two Classes of Principal Cells in the Mouse Piriform Cortex

The piriform (or primary olfactory) cortex is a trilaminar structure that is the first cortical destination of olfactory information, receiving monosynaptic input from the olfactory bulb. Here, we show that the main input layer of the piriform cortex, layer II, is dominated by two classes of principal neurons, superficial pyramidal (SP) and semilunar (SL) cells, with strikingly different properties. Action potentials in SP cells are followed by a Ni2+-sensitive afterdepolarization that promotes burst firing, whereas SL cells fire nonbursting action potentials that are followed by a powerful afterhyperpolarization. Synaptic inputs from the olfactory bulb onto SP cells exhibit prominent paired-pulse facilitation, which is attributable to residual presynaptic Ca2+ and a low probability of neurotransmitter release. In contrast, the same inputs onto SL cells do not facilitate. These distinctive synaptic and firing properties cause SP and SL cells to respond differently to in vivo-like bursts of afferent stimulation: SP cells tend to fire bursts of output action potentials at a higher frequency than the input, whereas SL cells tend to fire at a lower frequency than the input. When connected together in the canonical circuit of the piriform cortex, SP and SL cells transform the pattern of synaptic inputs they receive from the olfactory bulb, dispersing the firing rate and latency of output action potentials to an extent that depends on the strength of the input. Thus, the presence of two types of principal cells in layer II of the piriform cortex may underlie coding strategies used for the representation of odors.

Inhibitory Neurons in the Anterior Piriform Cortex of the Mouse: Classification Using Molecular Markers

The primary olfactory cortex (or piriform cortex, PC) is attracting increasing attention as a model system for the study of cortical sensory processing, yet little is known about inhibitory neurons in the PC. Here we provide the first systematic classification of GABA‐releasing interneurons in the anterior PC of mice, based on the expression of molecular markers. Our experiments used GAD67‐GFP transgenic mice, in which gamma‐aminobutyric acid (GABA)‐containing cells are labeled with green fluorescent protein (GFP). We first confirmed, using paired whole‐cell recordings, that GFP+ neurons in the anterior PC of GAD67‐GFP mice are functionally GABAergic. Next, we performed immunolabeling of GFP+ cells to quantify their expression of every possible pairwise combination of seven molecular markers: calbindin, calretinin, parvalbumin, cholecystokinin, neuropeptide Y, somatostatin, and vasoactive intestinal peptide. We found that six main categories of interneurons could be clearly distinguished in the anterior PC, based on the size and laminar location of their somata, intensity of GFP fluorescence, patterns of axonal projections, and expression of one or more of the seven markers. A number of rarer categories of interneurons could also be identified. These data provide a road map for further work that examines the functional properties of the six main classes of interneurons. Together, this information elucidates the cellular architecture of the PC and provides clues about the roles of GABAergic interneurons in olfactory processing. J. Comp. Neurol. 518:1670–1687, 2010.

Distinctive Classes of GABAergic Interneurons Provide Layer-Specific Phasic Inhibition in the Anterior Piriform Cortex

The primary olfactory (or piriform) cortex is a trilaminar paleocortex that is seen increasingly as an attractive model system for the study of cortical sensory processing. Recent findings highlight the importance of γ-amino butyric acid (GABA)-releasing interneurons for the function of the piriform cortex (PC), yet little is known about the different types of interneurons in the PC. Here, we provide the first detailed functional characterization of the major classes of GABAergic interneurons in the anterior piriform cortex (aPC) and show how these classes differentially engage in phasic synaptic inhibition. By measuring the electrical properties of interneurons and combining this with information about their morphology, laminar location, and expression of molecular markers, we have identified 5 major classes in the aPC of the mouse. Each layer contains at least one class of interneuron that is tuned to fire either earlier or later in a train of stimuli resembling the input received by the PC in vivo during olfaction. This suggests that the different subtypes of interneuron are specialized for providing synaptic inhibition at different phases of the sniff cycle. Thus, our results suggest mechanisms by which classes of interneurons play specific roles in the processing performed by the PC in order to recognize odors.

Quantal analysis of synaptic transmission in the central nervous system.

In the past year, important advances have been made in the understanding of quantal neurotransmission at central synapses. These include new statistical tests for the significance of quantal peaks in synaptic amplitude histograms, greater understanding of possible sources of quantal variance, and new attempts to undertake a rigorous quantal analysis of neurotransmission.